It has long been thought that suppressor cells play a role in the progression of cancer (Dye et al., J. Exp. Med. 154:1033-1042 (1981)). In fact, active suppression by T regulatory cells plays an important role in the down-regulation of T cell responses to foreign and self-antigens.
T cells are a class of lymphocytes, having specific T cell receptors (TCRs) that are produced as a result of gene rearrangement. T cells have diverse roles, which are accomplished by the differentiation of distinct subsets of T cells, recognizable by discrete patterns of gene expression. Several major T cell subsets are recognized based on receptor expression, such as TCR-α/β, and TCR γ/Δ and invariant natural killer cells. Other T cell subsets are defined by the surface molecules and cytokines secreted therefrom. For example, T helper cells (CD4 cells) secrete cytokines, and help B cells and cytotoxic T cells to survive and carry out effector functions. Cytotoxic T cells (CTLs) are generally CD8 cells, and they are specialized to kill target cells, such as infected cells or tumor cells. Natural killer (NK) cells are related to T cells, but do not have TCRs, and have a shorter lifespan, although they do share some functions with T cells and are able to secrete cytokines and kill some kinds of target cells.
Human and mouse peripheral blood contains a small population of T cell lymphocytes that express the T regulatory phenotype (“Treg”), i.e., positive for both CD4 and CD25 antigens (i.e., those CD4+ T cells that are also distinctly positive for CD25). First characterized in mice, where they constitute 6-10% of lymph node and splenic CD4+ T cell populations, this population of CD4+CD25+ cells represents approximately only 5-10% of human peripheral blood mononuclear cells (PBMC), or 2-7% of CD4+ T cells, although some donors exhibit a more distinct population of CD4+ and CD25+ cells. About 1-2% of human peripheral blood PBMCs are both CD4 positive (CD4+) and CD25 brightly positive (CD25) cells.
There are several subsets of Treg cells (Bluestone et al., Nature Rev. Immunol. 3:253 (2003)). One subset of regulatory cells develops in the thymus. Thymic derived Treg cells function by a cytokine-independent mechanism, which involves cell to cell contact (Shevach, Nature Rev. Immunol 2:389 (2002)). They are essential for the induction and maintenance of self-tolerance and for the prevention of autoimmunity (Shevach, Annu. Rev. Immunol. 18:423-449 (2000); Stephens et al., 2001; Taarns et al., 2001; Thornton et al., 1998; Salomon et al., Immunity 12:431-440 (2000); Sakaguchi et al., Immunol. Rev. 182:18-32 (2001)). These professional regulatory cells prevent the activation and proliferation of autoreactive T cells that have escaped thymic deletion or recognize extrathymic antigens, thus they are critical for homeostasis and immune regulation, as well as for protecting the host against the development of autoimmunity (Suri-Payer et al., J. Immunol. 157; 1799-1805 (1996); Asano et al., J. Exp. Med. 184:387-396 (1996); Bonomo et al., J. Immunol. 154:6602-6611 (1995); Willerford et al., Immunity 3:521-530 (1995); Takahashi et al., Int. Immunol. 10:1969-1980 (1998); Salomon et al., Immunity 12:431-440 (2000); Read et al., J. Exp. Med. 192:295-302 (2000). Thus, immune regulatory CD4+CD25+ T cells are often referred to as “professional suppressor cells,”
However, Treg cells can also be generated by the activation of mature, peripheral CD4+ T cells. Studies have indicated that peripherally derived Treg cells mediate their inhibitory activities by producing immunosuppressive cytokines, such as transforming growth factor-beta (TGF-β) and IL-10 (Kingsley et al., J. Immunol. 168:1080 (2002); Nakamura et al., J. Exp. Med. 194:629-644 (2001)). After antigen-specific activation, these Treg cells can non-specifically suppress proliferation of either CD4+ or CD25+ T cells (demonstrated by FACS sorting in low dose immobilized anti-CD3 mAb-based co-culture suppressor assays by Baecher-Allan et al., J. Immunol. 167(3):1245-1253 (2001)).
Studies have shown that CD4+CD25+ cells are able to inhibit anti-CD3 stimulation of T cells when co-cultured with autologous antigen presenting cells (APC), but only through direct contact (Stephens et al., Eur. J. Immunol. 31:1247-1254 (2001); Taams et al., Eur. J. Immunol. 31:1122-1131 (2001); Thornton et al., J. Exp. Med. 188:287-296 (1998)). However, in mice this inhibitory effect was not able to overcome direct T cell stimulation with immobilized anti-CD3 or with anti-CD3/CD28 (Thornton et al., 1998). In previous reports, human CD4+CD25+ T cells isolated from peripheral blood required pre-activation in order to reveal their suppressive properties, as direct culture of the regulatory cells was generally insufficient to mediate suppressive effects (Dieckmann et al., J. Exp. Med. 193:1303-1310 (2001)). Others have also found that the inhibitory properties of human CD4+CD25+ T cells are activation-dependent, but antigen-nonspecific (Jonuleit et al., J. Exp. Med. 193:1285-1294 (2001); Levings et al., J. Exp. Med. 193(11):1295-1302 (2001); Yamagiwa et al., J. Immunol. 166:7282-7289 (2001)), and have demonstrated constitutive expression of intracellular stores of cytotoxic T lymphocyte antigen-4 (CTLA-4) (Jonuleit et al., 2001; Read et al., J. Exp. Med. 192:295-'302 (2000); Yamagiwa et al., 2001; Takahashi et al., J. Exp. Med. 192:303-310 (2000)). Moreover, after T-Cell receptor (TCR)-mediated stimulation, CD4+CD25+ T cells suppress the activation of naive CD4+CD25+ T cells activated by alloantigens and mitogens (Jonuleit et al., 2001).
Both mouse and human Treg cells express CTLA-4, however the role of CTLA-4 in tolerance induction and its capacity to impart inhibitory function to regulatory CD4+CD25+ T cells is controversial. CTLA-4 (also known as CD152) is a homolog of CD28 and is a receptor for the CD80 and CD86 ligands. CTLA-4 inhibits T cell responses in an antigen and TCR-dependent manner. T cells that have impaired CTLA-4 function have enhanced T cell proliferation and cytokine production. In contrast, enhanced CTLA-4 function leads to inhibited cytokine secretion and impaired cell cycle progression both in vitro and in vivo. In the mouse, CTLA-4 is not required for suppressive function of the Treg cells, as opposed to its requirement in humans. This may be explained in part by the recent discovery that there are multiple forms of CTLA-4, and that this can vary between strains of mice or humans.
A recent study has shown that Treg cells grow extensively in vivo (Tang, J. Immunol. 171:3348 (2003)), while others have suggested that the efficacy of therapeutic cancer vaccination in mice can be enhanced by removing CD4+CD25+ T cells (Sutmuller et al., J. Exp. Med. 194:823-832 (2001)). Studies have also indicated that depletion of regulatory cells led to increased tumor-specific immune responses and eradication of tumors in otherwise non-responding animals (Onizuka et al., Cancer Res. 59:3128-3133 (1999); Shimizu et al., J. Immunol. 163:5211-5218 (1999)). Susceptible mouse strains that were made CD4+CD25+ deficient by neonatal thymectomy were shown to develop a wide spectrum of organ-specific autoimmunities that could be prevented by an infusion of CD4+CD25+ T cells by 10-14 days of ago (Suri-Payer et al., J. Immunol. 160:1212-1218 (1998)). That study also found that CD4+CD25+ T cells could inhibit autoimmunity induced by autoantigen-specific T cell clones. The transfer of CD4+CD25− T cells into nude mice also reportedly led to the development of autoimmune disorders which could be prevented by the co-transfer of CD4+CD25+ T cells using lymphocytes first depleted of CD25+ cells (Sakaguchi et al., J. Immunol. 155:1151-1164 (1995)).
However, data also indicate that the role of CD4+CD25+ cells is not limited to self-tolerance and the prevention of autoimmunity. While few studies have addressed the role of CD4+CD25+ T cells in alloresponses or in transplantation, CD4+CD25+ T cells have been reported to prevent allograft rejection, both in vitro and in vivo (Hara et al., J. Immunol. 166:3789-3796 (2001); Taylor et al., J. Exp. Med. 193:1311-1318 (2001)). Allogeneic stimulation of human T cell proliferation is also blocked by CD4+CD25+ T cells (Yamagiwa et al., 2001), whereas Wood's laboratory has shown that CD4+CD25+ T cells suppress mixed lymphocyte responses (MLR), but only when the alloantigen was presented by the indirect, and not the direct, pathway of allorecognition (Hara et al., 2001). It is likely that direct antigen presentation occurs between the regulatory T cells and the anti-CD3/28 stimulated responder T cells, as the sorted CD4+25+ cells are highly depleted of professional APC.
The inventors have shown that CD4+CD25+ T cells exist in high proportions in the turner infiltrating lymphocytes of patients with non-small cell lung cancer (NSCLC) (Woo et al., Cancer Res. 61:4766-4772 (2001)), and that CD4+CD25+ cells were an essential requirement for the ex vivo induction of tolerance to alloantigen via co-stimulatory blockade (Taylor et al., J. Exp. Med. 193:1311-1318 (2001)). Most of the literature states, however, that the immune system is in a state of ignorance to peripheral solid tumors, thus it is anergic (Ochsenbein, et al., Nature 411:1058-1064 (2001); Staveley-O'Carroll et al., Proc. Natl. Acad. Sci. USA 95:1178-1183 (1998)). The explanation for the differential ability of the CD4+CD25+ T cells to suppress autologous and allogeneic T cell proliferation is most likely complex. As a result, the role of CD4+CD25+ T cells in human tumors or any effect that they may have in preventing the host from mounting an immune response to autoantigens, such as tumor antigens, has to date remained unknown.
Treg are have been described in the literature as being hypoproliferative in vitro (Sakaguchi, Ann. Rev. Immunol. 22:531 (2004)). Trenado et al. provided the first evaluation of the therapeutic efficacy of ex vivo activated and expanded CD4+CD25+ regulatory cells in an in vivo animal model of disease (Trenado et al., J. Clin. Invest. 112(11):1688-1696 (2002)). In that situation, the infusion of ex vivo activated and expanded donor CD4+CD25+ cells was shown to significantly inhibit, rapidly-lethal GVHD, however, these data are presented only for mice—not in humans. Moreover, in the murine studies for the conditions tested, although the freshly isolated or cultured Treg cells have been able to suppress GVHD, graft-versus-leukemia effects (GVL activity) was allowed (Trenado et al., 2002; Jones et al., Biol. Blood Marrow Transplant 9(4):243-256 (2003); Edinger et al., Nat. Med. 9(9):1144-1150 (2003)), as was immune reconstitution (Trenado et al., 2002).
However, human blood is quite different in composition from that of a mouse, meaning that without extensive experimentation, the murine studies cannot be translated into equivalent responses in human cells. Human blood contains memory cells (˜50%), which may be CD25 dim and overlap with the CD4+CD25+ suppressor cell population, making human Treg cells very difficult to purify. By comparison, the CD25 dim cells are only minimally present in rodents, or completely absent from young pathogen free mice (the condition utilized in most murine studies). In humans, purification of Treg based on CD25-selection (the only known surface marker of circulating suppressor cells, since CTLA-4 is not on the surface of fresh cells) results in enrichment of Treg cells, but it is not sufficient for full purification. Partially pure suppressor populations may briefly evidence suppression following short-term culture/activation, but these are quickly overgrown by the contaminating conventional T cells.
As a result, findings comparable to those of Trenado et al. have never been reported for human cells. Those published reports that do show proliferation of CD4+CD25+ cells, fail to find suppressor function, and until the present invention, no one has been able to obtain extensive in vitro or ex vivo expansion of human Treg cells, while at the same time maintaining GMP conditions. Only one prior publication describes the expansion of human CD4+CD25+ cells (Levings et al., 2001). Yet, in this paper, only one figure is shown of suppressor function, and it is shown to have only a modest effect. With a 1:1 ratio of suppressor cells to responder cells, only approximately 60%-65% inhibition of proliferation was noted, which is less than that which is typically observed with mouse Treg cells. Thus, the reported suppression was of such a small magnitude, that is could have resulted from non-specific effects (e.g., growth factor consumption, overcrowding, displacement from antigen presenting cells etc). Moreover, the culture was maintained by Levings et al. for only a short term (only 14 days), and the cells most likely represent a mixed culture of regulatory cells and conventional T cells.
To culture the Treg cells, Leavings et al. used JY lymphoblastoid cells (BBV virally transformed lymphoblastoid cell line) cultured with soluble anti-CD3 (1 μg/ml), in the presence of a feeder cell mixture of allogeneic PBMCs. Purification of the CD4+CD25+ cells was reported by the authors using a two stage magnetic microbead protocol, wherein first the cells were depleted of non-T cells and CD8 type of T cells using antibodies to CD8, CD11b, CD16, CD19, CD36, and CD56, which makes the resulting product unsuitable for therapeutic use in humans. Then, the cells were selected for CD25 positivity.
Yet, while Leavings et al. reported 90% purity of the Treg cells, no disclosure was made regarding stringency. This is problematic, since a very high level of stringency is absolutely critical for the isolation of human cells of sufficient purity (CD25+) for suppressor cell line generation, a finding that until the present invention, has neither been, discussed nor appreciated in the prior art. However, as will be shown below, the inadequacy of isolation and expansion methods used by others for the generation of the suppressor cell lines, has significantly interfered with advances in the research on human Treg cells. Consequently, it has not been possible to previously use Treg cells effectively for therapeutic purposes.
Thus, there has been a need for methods of producing sufficient number of these Treg cells to permit characterization and to provide for safe and effective therapeutic use in human patients. There has also remained a need for a greater understanding of the CD4+CD25+ T cells and their function in tumor immuno-surveillance and in the immunotherapy or immunosuppression of cancers, particularly solid tumor cancers, such as lung cancer. Equally important has been a need to suppress in vivo alloresponses and autoimmune responses, such as, although not limited to, graft-vs-host disease (GVHD), and to elucidate and expand upon the role of CD4+CD25+ cellular therapy and to define methods for isolating or producing such CD4+CD25+ suppressor cells.